Dysresgulated metabolic pathways contribute to several diseases, including cancer. The discovery and functional characterization of biochemical pathways that support cancer have, however, been hindered by a lack of technologies that can broadly profile metabolites, enzymes, and metabolite- protein interactions in native biological systems. We have addressed this central problem through the development and application of an innovative set of chemical proteomic and metabolomic technologies. In this competitive renewal application, we propose to use and expand our chemical proteomic and metabolomic methods towards the two major goals of: 1) characterizing metabolic (de)methylation pathways that support the growth and malignant properties of cancer cells, and 2) globally mapping and characterizing metabolite-protein interactions that support the growth and malignant properties of cancer cells. These studies are designed to test three major hypotheses: 1) dysregulated metabolic enzymes coordinately control the methylation potential, epigenetic state, and pro-tumorigenic properties of cancer cells, 2) photoreactive, clickable probes offer a general chemoproteomic strategy to map metabolite-protein interactions in living cells, and 3) mapping the full complement of proteins that bind to bioactive sterols and lipids in tumor cells will uncover new biochemical nodes of regulation and crosstalk that define metabolic dependencies of cancers. The ultimate goal of this application is to identify and functionally characterize metabolic pathways that are dysregulated in cancer and support tumorigenesis. The molecular components of these pathways may, in turn, represent new biomarkers and drug targets for the diagnosis and treatment of cancer. The research tools and methods advanced in this proposal should also prove of general value for characterizing biochemical networks in a wide range of physiological and disease processes.